13 C-Metabolic flux analysis ( 13 C-MFA) has greatly contributed to our understanding of plant metabolic regulation. However, the generation of detailed in vivo flux maps remains a major challenge. Flux investigations based on nuclear magnetic resonance have resolved small networks with high accuracy. Mass spectrometry (MS) approaches have broader potential, but have hitherto been limited in their power to deduce flux information due to lack of atomic level position information. Herein we established a gas chromatography (GC) coupled to MS-based approach that provides 13 C-positional labelling information in glucose, malate and glutamate (Glu). A map of electron impact (EI)-mediated MS fragmentation was created and validated by 13 Cpositionally labelled references via GC-EI-MS and GC-atmospheric pressure chemical ionization-MS technologies. The power of the approach was revealed by analysing previous 13 C-MFA data from leaves and guard cells, and 13 C-HCO 3 labelling of guard cells harvested in the dark and after the dark-to-light transition. We demonstrated that the approach is applicable to established GC-EI-MS-based 13 C-MFA without the need for experimental adjustment, but will benefit in the future from paired analyses by the two GC-MS platforms. We identified specific glucose carbon atoms that are preferentially labelled by photosynthesis and gluconeogenesis, and provide an approach to investigate the phosphoenolpyruvate carboxylase (PEPc)derived 13 C-incorporation into malate and Glu. Our results suggest that gluconeogenesis and the PEPcmediated CO 2 assimilation into malate are activated in a light-independent manner in guard cells. We further highlight that the fluxes from glycolysis and PEPc toward Glu are restricted by the mitochondrial thioredoxin system in illuminated leaves.

No abstract

Recent results suggest that metabolism‐mediated stomatal closure mechanisms are important to regulate differentially the stomatal speediness between ferns and angiosperms. However, evidence directly linking mesophyll metabolism and the slower stomatal conductance (gs) in ferns is missing. Here, we investigated the effect of exogenous application of abscisic acid (ABA), sucrose and mannitol on stomatal kinetics and carried out a metabolic fingerprinting analysis of ferns and angiosperms leaves harvested throughout a diel course. Fern stomata did not respond to ABA in the time period analysed. No differences in the relative decrease in gs was observed between ferns and the angiosperm following provision of sucrose or mannitol. However, ferns have slower gs responses to these compounds than angiosperms. Metabolomics analysis highlights that ferns have a higher accumulation of secondary rather than primary metabolites throughout the diel course, with the opposite being observed in angiosperms. Our results indicate that metabolism‐mediated stomatal closure mechanisms underpin the differential stomatal speediness regulation among ferns and angiosperms, in which the slower stomatal closure in ferns is associated with the lack of ABA‐responsiveness, to a reduced capacity to respond to mesophyll‐derived sucrose and to a higher carbon allocation toward secondary metabolism, which likely modulates both photosynthesis‐gs and growth‐stress tolerance trade‐offs.

No abstract

Water deficit (WD) combined with high temperature (HT) is the major factor limiting agriculture worldwide, and it is predicted to become worse according to the current climate change scenario. It is thus important to understand how current cultivated crops respond to these stress conditions. Here we investigated how four soybean cultivars respond to WD and HT isolated or in combination at metabolic, physiological, and anatomical levels. The WD + HT increased the level of stress in soybean plants when compared to plants under well-watered (WW), WD, or HT conditions. WD + HT exacerbates the increases in ascorbate peroxidase activity, which was associated with the greater photosynthetic rate in two cultivars under WD + HT. The metabolic responses to WD + HT diverge substantially from plants under WW, WD, or HT conditions. Myo-inositol and maltose were identified as WD + HT biomarkers and were connected to subnetworks composed of catalase, amino acids, and both root and leaf osmotic potentials. Correlation-based network analyses highlight that the network heterogeneity increased and a higher integration among metabolic, physiological, and morphological nodes is observed under stress conditions. Beyond unveiling biochemical and metabolic WD + HT biomarkers, our results collectively highlight that the mechanisms behind the acclimation to WD + HT cannot be understood by investigating WD or HT stress separately.